1. Field of the Invention
The invention relates to the field of cellular proteins, most particularly cellular proteins capable of binding nucleic acids and regulating gene expression. In addition, the invention also relates to the field of reagents useful in the characterization of viral and cellular gene expression, as the disclosed cellular protein is demonstrated to bind to a particular viral RNA region important in viral gene expression. The invention also relates to the field of therapeutic methods and reagents for the treatment of viral diseases, such as AIDS. Methods and reagents (including monoclonal antibodies) for screening/detecting viral infection in a biological sample are also within the field of the present invention.
2. Background of the Related Art
Gene expression of the human immunodeficiency virus (HIV) is regulated by a variety of mechanisms. The long terminal repeat (LTR) is the site of multiple regulatory regions involved in both general and tissue specific gene expression..sup.1-16 Two regions, including the NFAT.sup.17,18 and NF-kappa B motifs.sup.4,19,20 are involved in the regulation of the HIV LTR in activated T-lymphocytes. Other elements such as SPI.sup.2,11 and TATA.sup.3,7,10 are involved in regulating gene expression in a variety of both lymphoid and nonlymphoid cell lines.
HIV contains an additional regulatory element known as the transactivating region, TAR, which extends from -17 to +80 in the HIV LTR..sup.1,3,5-7
The TAR element is required for activation of gene expression by the viral transactivator protein, tat..sup.9,12,20-25 The structural integrity of TAR RNA is a key element for tat activation..sup.5,12,6,9 TAR RNA is capable of forming a stable stem-loop structure and disruption of stem base pairing results in a marked decrease in tat activation. However, compensatory mutations which restore stem base pairing result in nearly wild-type levels of tat activation..sup.5,6,15 The loop and bulge regions in TAR are also required for high level activation by tat..sup.5,13,27 Substitution of single base pairs in the loop decreases tat activation while substitution of multiple base pairs in this region result in even further decreases in tat activation..sup.9,10,15 Deletion of the bulge region, or substitution for a single "U" residue at +23 in the bulge, also severely decreases tat activation..sup.15,16,27 Thus, three major determinants including the stem, loop, and bulge are each required for wild-type activation of the TAR element and ultimate gene expression of HIV.
Recently, a number of studies indicate that the tat protein, via its basic domain, is capable of binding to the bulge region in TAR RNA..sup.16,27-29 A variety of nuclear proteins are also capable of binding to TAR RNA..sup.30-32 However, the role or importance of these "binding" proteins, particularly for the regulation of gene expression, has not been fully characterized.
A previous study used UV-crosslinking assays to identify a 68 kDa cellular protein that binds specifically to the TAR loop region..sup.26 Using similar techniques, the present inventors also detected a 68 kDa cellular protein which binds to TAR RNA. The detection of this protein was dependent on the use of heparin and ribonuclease following the binding reactions. However, when nonspecific RNA or poly (I)-poly (C) was used in gel retardation assays without ribonuclease, this species was no longer detected (unpublished results). Another RNA binding protein, TRP-140, which binds with high affinity to a variety of double-stranded RNAs, may also potentially have functional significance in regulating HIV gene expression.
Several viruses, such as those of the HIV and the HTLV (human leukemia/lymphoma virus) type, have within their gene structure a downstream regulatory region that is required for transactivation (a protein that acts in conjunction with a viral protein as a transactivating factor--the pX region.sup.49) to which a cellular protein binds. A more complete understanding of the mechanisms which govern viral gene expression, most particularly the role of particular binding proteins for the TAR region in expression, would provide a route whereby methods for selectively "turning on" and "turning off" of genes employing important cellular proteins could be accomplished. Most importantly, in the case of viral infections such as AIDS, such information could be employed to develop a therapeutic agent which selectively "turned off" the expression of HIV and HTLV in an animal.
HIV is recognized as the causative agent of Acquired Immunodeficiency Syndrome (AIDS). Therapeutic agents which have been used in the treatment of AIDS include AZT (azidothymidine) and DDI (dideoxyinosine)..sup.33 Both of these agents are nucleotide analogs that target the viral enzyme, reverse transcriptase. While these agents have been used with varying degrees of success, they are also unfortunately associated with a variety of severe side effects. Some of these side effects include peripheral neuropathy (DDI), pancreatitis, granulocytopenia, anemia, severe headache, nausea, insomnia, neurotoxicity, seizure, as well as being associated with a potential carcinogenicity and teratogenicity..sup.33
Other molecular targets for anti-viral therapy under investigation include an HIV-gene encoded protease. The protease is encoded on the polygene of HIV-1. The polygene encodes three proteins--a reverse transcriptase, a self-cleaving protease that is required for processing the reverse transcriptase, and a nuclease that is essential for integration of viral DNA into the genome of the host cell. Inhibitors of the HIV protease have been developed with the aid of a crystal structure of the protein.
Other potential molecular targets described include the glycosolated envelope protein of HIV and the receptor protein (CD4) on the surface of lymphocytes to which the virus binds. A soluble form of CD4 can bind to the viral envelope protein and prevent the virus from entering cells. Alternatively, a conjugate of CD4 and a toxin might be used to attack HIV-infected cells, since such cells express the envelope protein on their surfaces. Another drug, dextran sulfate, has also been used in the treatment of AIDS for its ability to block the binding of HIV to target cells.
However, none of the aforedescribed molecular targets for anti-viral therapy relates to use of a molecular agent of cellular origin which specifically affects viral gene expression. An enhanced understanding of the particular role of cellular proteins in the molecular events of both cellular and vital (HIV) gene expression would provide a new avenue for the development of effective anti-viral agents. Such information would further provide for the development of a new genus of drugs based on the regulation of host proteins for the treatment of diseases such as AIDS and AIDS-related diseases.
The present inventors have characterized a particular cellular protein designated "TRP-185", which possesses botch high affinity and marked specificity of binding to a viral TAR RNA region in a manner that correlates with in vivo genetic data in that the TRP-185 cellular protein only binds to templates that are activated by the transactivating protein.
Thus, a solution to providing for the specific inhibition activation of viruses, such as HIV and HTLV, which have a downstream binding region for important cellular proteins important in gene activation is disclosed.
It is an object of the invention to provide a tool which is useful in the characterization of viral and cellular gene expression. It is still another object of the invention to provide a reagent which is useful in the study of viral gene regulation.
It is still a further object of this invention to provide a better, more reliable and convenient procedure for testing human serum and plasma for presence of the AIDS virus.
It is another object of the invention to provide a method for monitoring the progression and treating HIV-disease in a patient. How these and other objects of this invention are achieved will become apparent in light of the accompanying disclosure. In at least one embodiment of the practices of this invention, at least one of the foregoing objects will be achieved.